Novel proteins within the type e botulinum neurotoxin complex

ABSTRACT

The invention features a polypeptide complex synthesized by bacteria of the genus  Clostridia  that contains the serotype E botulinum neurotoxin and five neurotoxin associated polypeptides having molecular weights of about 118, 80, 65, 40, and 18 kDa, respectively. The complex is useful in the treatment of diseases or conditions that are caused by excessive release of acetylcholine from presynaptic nerve terminals.

This application is a continuation of U.S. application Ser. No.12/247,124, filed on Oct. 7, 2008, now U.S. Pat. No. 7,981,432, which isa divisional of U.S. application Ser. No. 10/766,283, filed on Jan. 27,2004, now U.S. Pat. No. 7,431,935, which is a divisional of U.S.application Ser. No. 09/546,136, filed on Apr. 10, 2000, now U.S. Pat.No. 6,699,966, which is a continuation of U.S. application Ser. No.08/889,354, filed on Jul. 8, 1997, now abandoned, which claims thebenefit of priority from U.S. Provisional Application No. 60/021,348,filed on Jul. 8, 1996, which are all incorporated herein by reference intheir entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This work was supported by grants from the United States Department ofAgriculture (94-37201-1167) and the United States Department of Defense(NRL-ERDC-N00014-92-K-2007). The U.S. government may have certain rightsin the invention.

BACKGROUND OF THE INVENTION

The present invention relates to novel proteins that form a complex withthe type E botulin neurotoxin.

Various strains of the bacterium Clostridium, including C. botulinum, C.baratii, and C. butyricum, synthesize different serotypes of the potentneurotoxin botulin, which causes a form of food poisoning known asbotulism. C. botulinum synthesizes seven different serotypes of botulin,which are designated by the letters A through G. Humans and otheranimals come into contact with these neurotoxins most frequently byconsuming food that is improperly stored in a way that permits growth ofanaerobic bacteria. Foods that are typically tainted with botulininclude low acid canned meats and vegetables, preserved meats and fish,and pasteurized processed cheese spreads (Fogeding, In FoodborneMicroorganisms and Toxins: Developing Methodology, M. D. Pierson and N.Sterns, Eds., Marcel Dekker, Inc., New York, N.Y., 1986; Kautter et al.,J. Food Prot. 42:784-786, 1979).

Another form of botulism, infant botulism, is thought to be caused byconsumption of ubiquitous spores of C. botulinum along with food(Simpson, In Botulinum Neurotoxin and Tetanus Toxin, Academic Press, SanDiego, Calif., 1989). These spores may colonize the infant intestine,germinate, and produce the neurotoxin. Similarly, spores that gainaccess to deeply wounded tissue may germinate and produce neurotoxinwithin the wound.

Once present in the body, botulin neurotoxins cause muscle paralysis byblocking the release of acetylcholine from cholinergic nerve endings(DasGupta et al., Biochemistry and Pharmacology of Botulinum and TetanusNeurotoxins, In Perspective in Toxicology, A. W. Bernheimer, Ed., Wiley,New York, N.Y., 1977). Death may be caused by paralysis of therespiratory muscles.

The nucleotide sequences of the genes encoding all of the differentserotypes of the neurotoxin are known (Binz et al., J. Biol. Chem.265:9153-9158, 1990; Campbell et al., J. Clin. Microbiol. 31:2255-2262,1993; East et al., FEMS Microbiol. Lett. 96:225-230, 1992; Hauser etal., Nucl. Acids Res. 18:4924, 1990; Whelan et al., Eur. J. Biochem.,204:657-667, 1992; and Whelan et al., Appl. Environ. Microbiol.58:2345-2354, 1992). These genes are coordinately regulated with thoseencoding proteins that form complexes with the various serotypes ofbotulin (Fujii et al., J. Gen. Microbiol. 139:79-83, 1993; and Nukina etal., In Botulinum and Tetanus Neurotoxins, B. R. DasGupta, Ed., PlenumPress, New York, N.Y., 1993). The A and B type neurotoxins areassociated with at least five other proteins, called “neurotoxin bindingproteins” or NAPS, while the type E neurotoxin has been found inassociation with only one other protein (Sugii et al., Infect. Immunol.12:1262-1270, 1975; Sakaguchi, Pharmac. Ther. 19:165-194, 1983; Schantzet al., Microbiol. Rev. 56:80-99, 1992; and Singh et al., J. ProteinChem. 14:7-18, 1995).

The proteins that associate with the type A neurotoxin play a criticalrole in the food poisoning process by protecting the neurotoxin from theacids and proteolytic enzymes present in the gastrointestinal tract. Forexample, it is known that the oral toxicity of the intact type Aneurotoxin complex is 43,000 times greater than the oral toxicity ofisolated and purified type A neurotoxin (Sakaguchi, Pharmac. Ther.19:165-194, 1983). The proteins associated with other serotypessimilarly “protect” the neurotoxin, but to a lesser degree.

SUMMARY OF THE INVENTION

The invention is based on the discovery that the type E botulinum toxinexists in a complex that includes the toxin and five other polypeptidestermed neurotoxin associated proteins (NAPs). This discovery is contraryto the prior assertions of those in the field, who believed that thetoxin was associated with only one other polypeptide, a neurotoxinbinding protein (NBP) having a molecular weight of approximately 118kDa.

Accordingly, the invention features a substantially pure polypeptidecomplex that includes a Clostridium botulinum neurotoxin and more thanone Clostridium botulinum type E neurotoxin associated polypeptide. Thepolypeptides of the invention include the newly discovered NAPs, whichhave molecular weights of approximately 80, 65, 40, and 18 kDa, andsubstantially pure antibodies that specifically bind to one or more ofthese polypeptides, or complexes of one or more of the NAPs with type Ebotulinum toxin, or toxins from other Clostridium botulinum serotypesincluding A, B, C, D, F, and G.

The following peptide sequences have been obtained: (1) MKQAFVFEFD (SEQID NO:1), from the 18 kDa polypeptide; (2) MRINTNINSM (SEQ ID NO:2),from the 40 kDa polypeptide; (3) MQTTTLNWDT (SEQ ID NO:3), from the 65kDa polypeptide; and (4) TNLKPYIIYD (SEQ ID NO:4), from the 80 kDapolypeptide. In addition, the complete amino acid sequence of the 18 kDapolypeptide has been obtained and is shown in FIG. 8 (SEQ ID NO:5).

The compositions of the invention (e.g., the novel NAPs and antibodiesthat specifically bind to them) can be used to detect the serotype Eneurotoxin complex in a sample. For example, one can contact the samplewith an antibody that specifically binds a NAP, or with a NAP that bindsthe neurotoxin itself (as described in the Examples, below, the NAPhaving an approximate molecular weight of 80 kDa binds directly to thetype E neurotoxin) and detect, if present, antibody-bound type Eassociated polypeptide or NAP-bound type E neurotoxin. The presence ofthese antibody complexes indicates the presence of serotype E neurotoxinin the sample. The detection methods of the invention can be used toexamine virtually any type of sample, including samples of foodstuffs,or biological samples, such as gastrointestinal, blood, or tissuesamples obtained from a vertebrate animal.

The novel compositions of the invention also provide the basis formethods of treating patients who suffer from a disease or conditionsassociated with excessive release of acetylcholine from presynapticnerve terminals. The patient is treated by administration of atherapeutically effective amount of a polypeptide complex that containsthe serotype E neurotoxin (or other serotype toxin) and more than oneNAP, e.g., one or more of the 80, 65, 40, and 18 kDa NAPs, and/or the118 kDa polypeptide. The conditions associated with excessiveacetylcholine release include undesirable contractions of smooth orskeletal muscle cells, which can, in turn, cause spasmodic torticollis,essential tremor, spasmodic dysphonia, charley horse, strabismus,blepharospasm, oromandibular dystonia, spasms of the sphincters of thecardiovascular, gastrointestinal, or urinary systems, or tardivedyskinesia. Excessive release of acetylcholine can also cause profusesweating, lacrimation, or mucous secretion. Patients who could benefitfrom the methods described herein include those who suffer fromspasticity that occurs secondary to another event such as brainischemia, traumatic injury of the brain or spinal cord, tensionheadache, or pain (e.g., pain caused by sporting injuries or arthritis).

In addition, the novel compositions of the invention can be formulatedas a vaccine and administered to an animal in an amount sufficient toconfer a degree of protection against serotype E (or other) neurotoxin.

Administration of a purified neurotoxin complex (e.g., of the type E (orother) neurotoxin and more than one of the new NAPs), as describedbelow, is superior to administration of the neurotoxin alone because,within the complex, the neurotoxin is more stable and thus,longer-lasting. This feature minimizes the frequency of administrationand thereby reduces any risk, discomfort, or inconvenience that thepatient may experience.

The type E complex is a superior therapeutic agent, relative to theother botulinum serotypes, because the activity of the type E neurotoxincan be enhanced 100-fold by treatment with trypsin, which breaks thebonds between the two polypeptide chains that constitute the neurotoxin.Therefore, application of the type E neurotoxin complex can becontrolled by trypsinization, in a way that allows graded release of theneurotoxin from the complex. This unique mechanism provides morecontrolled and longer-lasting effects than would otherwise be possible.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an elution profile obtained by applying an extract of E-typeproducing C. botulinum to a DEAE-Sephadex A-50 ion-exchange column (A278is absorbance at 278 nm).

FIG. 2 is a photograph of a polyacrylamide gel. Lane 1 was loaded withthe material that eluted in the first peak of FIG. 1. Lane 2 was loadedwith molecular weight standards. Lane 3 was loaded with material elutedfrom a G-200 column (see FIG. 5).

FIG. 3 is an elution profile obtained by applying the type E botulinumneurotoxin complex to a Sephadex G-100 column.

FIG. 4 is an elution profile obtained by applying the type E botulinumneurotoxin complex eluted from a Sephadex G-100 column to a SephadexG-200 column.

FIG. 5 is an elution profile of the complex formed between type Ebotulinum neurotoxin and the 80 kDa component of the associated proteincomplex.

FIG. 6 is a photograph of an SDS-polyacrylamide gel. The material in thefirst and second peaks of the elution profile shown in FIG. 5 waselectrophoresed in lanes 1 and 2, respectively.

FIG. 7 is a three-dimensional plot generated by light scatteringanalysis of the type E botulinum complex. The first and second series ofpeaks were generated with 105 nm and 225 nm diameter particles,respectively.

FIG. 8 is a representation of the amino acid sequence of a type Ebotulin NAP having a molecular weight of approximately 18 kDa (SEQ IDNO:5).

FIG. 9 is a schematic diagram of the genomic organization of C.botulinum neurotoxin serotypes C, A and B, F, and E.

DETAILED DESCRIPTION

Contrary to the general understanding in the field, the type E botulinumtoxin complex consists in vivo of the neurotoxin, which has a molecularweight of about 150 kDa, and five (not one, as previously believed)polypeptides that form a complex with the neurotoxin. These fivepolypeptides have molecular weights of approximately 118, 80, 65, 40,and 18 kDa. Those of skill in the art will recognize that the measuredor apparent molecular weight of a polypeptide can vary depending, forexample, on the number of glycosylated residues, and even on the methodused to determine the molecular weight. Accordingly, the measuredmolecular weights of the NAPs of the invention can vary. For example,the measured molecular weight of the approximately 80 kDa polypeptidecan vary between 70 and 90 kDa; the measured molecular weight of theapproximately 65 kDa polypeptide can vary between 60 and 70 kDa; themeasured molecular weight of the approximately 40 kDa polypeptide canvary between 35 and 45 kDa; and the measured molecular weight of theapproximately 18 kDa polypeptide can vary between 15 and 21 kDa.

The novel polypeptides discovered in the type E botulinum neurotoxincomplex can be used in a number of ways. First, they can be used todetect the presence of the type E botulin toxin in a sample, such as afood sample or a sample of biological tissue, or to generate antibodiesthat can be used in analogous detection methods. For example, if apatient is exposed to the neurotoxin, the NAPs and NAP-bindingantibodies of the invention provide the means (through direct bindingdetection systems or antibody-based detection systems) for rapid andreliable diagnosis. The NAPs, in their naturally occurring complex withthe type E neurotoxin (or complexed individually or in groups with typeE or other neurotoxins), are also useful in treating diseases associatedwith excessive release of acetylcholine from cholinergic nerve terminalsand, in addition, they can be used to generate vaccines forimmunization. These uses are described in further detail below.

Polypeptides of the Invention

The polypeptides of the invention include NAPs having molecular weightsof approximately 80, 65, 40, and 18 kDa (alone or in any combination,particularly in a combination that includes the type E (or other)botulin neurotoxin, and/or the neurotoxin binding protein (NBP) having amolecular weight of approximately 118 kDa), and antibodies thatspecifically bind to one or more of these NAPs or NAP complexes.

The invention encompasses full-length NAPs as well as fragments thatcorrespond to functional domains of the NAPs (e.g., fragments that bindto polypeptides in the type E botulin complex and help to increase thestability of the neurotoxin in vitro or in vivo, or fragments that areantigenic (i.e., that elicit the production of antibodies)). The NAPs ofthe invention, and fragments thereof, can have the sequence of awild-type NAP, or can contain a mutation (including deletions,additions, or substitutions of one or more amino acid residues).Preferably, the mutant polypeptides retain at least 50%, 75%, or atleast 95% or more of the biological activity of the correspondingwild-type polypeptide. It is a straightforward matter to compare thebiological activities of the mutant and wild-type polypeptides. Theycan, for example, be used in side-by-side tests for lethality inrodents. If an equivalent number of animals are killed regardless ofwhether they receive a particular dose of a type E complex containingwild-type or mutant NAPs, the mutant NAPs would be said to retain thebiological activity of the wild-type NAPs. If only half as many animalsdie following administration of a complex containing a mutant NAP, thenthe mutant would be said to retain half the biological activity of thewild-type NAP. Those of skill in the art will be aware of numerous waysin which biological activities can be fairly compared.

Mutant NAPs that contain a substitution of one or more amino acidresidues can be made purposefully or randomly (e.g., by using routinetechniques of recombinant DNA engineering or random mutagenesis,respectively). Amino acid substitutions may be purposefully changed toaffect the polarity, charge, solubility, hydrophobicity, hydrophilicity,or amphipathic nature of the residues involved. Not only may the mutantpolypeptides retain biological activity, this activity could beincreased. For example, a mutant NAP could be made to bind with a higheraffinity to the type E botulin neurotoxin.

Polypeptides in which the NAPs are fused to an unrelated protein (e.g.,a protein that can be easily detected or quantitated) are alsoconsidered within the scope of the invention.

The polypeptides of the invention can be purified from anaturally-occurring source, chemically synthesized (e.g., see Creighton,In Proteins: Structures and Molecular Principles, W.H. Freeman & Co.,New York, N.Y., 1983), or produced by recombinant DNA technology usingtechniques well known in the art for expressing nucleic acids. Thesemethods can, for example, be used to construct expression vectorscontaining a NAP-encoding sequence, and appropriate transcriptional andtranslational control signals.

“Substantially pure” polypeptides are polypeptides in preparations inwhich they represent at least 60% by weight of the preparation. When oneor more NAPs are present in a complex, for example, in a complex withthe type E botulin neurotoxin, a substantially pure preparation willconsist of at least 60%, by weight, the polypeptides of the givencomplex. Preferably, preparations containing one or more NAPs are atleast 75%, at least 90%, or even at least 99%, by weight, thepolypeptide(s) of interest. Purity can be readily measured by anyappropriate method, for example, column chromatography, polyacrylamidegel electrophoresis, or HPLC analysis.

The amino acid sequence of the NAP having a molecular weight ofapproximately 18 kDa has been determined (FIG. 8; SEQ ID NO:5), andpartial sequences are described herein for the NAPs having molecularweights of approximately 80 (SEQ ID NO:4), 65 (SEQ ID NO:3), and 40 kDa(SEQ ID NO:2). It is well within the abilities of those of ordinaryskill in the art to obtain additional sequence information from thepartial sequences disclosed herein. For example, PCR technology can beused to isolate full length NAP cDNA sequences as follows. First, RNAcan be isolated, following standard procedures, from an appropriatecellular or tissue source (e.g., a bacterium of the genus Clostridia),and reverse transcribed using an oligonucleotide primer specific for themost 5′ end of the amplified fragment. This oligonucleotide primes firststrand synthesis. The resulting RNA/DNA hybrid can then be “tailed” withguanines using a standard terminal transferase reaction, and the hybridcan be digested with RNAse H. Second strand synthesis can then be primedwith a poly-C primer. The sequence of the amplified fragment can then beobtained using any number of routine procedures.

Production of Antibodies Against the Type E Neurotoxin AssociatedProteins

Antibodies that specifically bind to one or more epitopes of a NAP, orfragments or derivatives thereof, are also considered within the scopeof the present invention. An antibody is said to “specifically bind” toa polypeptide when it recognizes and binds to that polypeptide, but notto other molecules in a sample (e.g., a biological sample that includestype E neurotoxin associated polypeptides). The antibodies of theinvention can be polyclonal, monoclonal, humanized, chimeric, or singlechain antibodies, or Fab fragments, F(ab′)2 fragments, fragmentsproduced by a Fab expression library, anti-idiotype (anti-Id)antibodies, and epitope-binding fragments of any of the types ofantibodies listed above.

A variety of standard methods can be used to generate antibodies againstthe type E neurotoxin associated proteins. For example, the type Eneurotoxin associated proteins, either individually or in theircomplexed forms, can be administered to an animal, such as a rat, mouse,or rabbit, to induce the production of polyclonal antibodies.Alternatively, antigenic fragments of the individual polypeptides can beused to generate polyclonal antibodies. Various adjuvants can be used toincrease the immunological response to an antigen, depending on the hostspecies. These adjuvants include Freund's (complete or incomplete)adjuvant, mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Potentiallyuseful human adjuvants are known to include BCG (bacile Calmette-Guerin)and Corynebacterium parvum.

In addition, antibodies according to the invention can be monoclonalantibodies (i.e., antibodies from a homogenous population that recognizea particular antigen) that are generated by using either individualserotype E NAPs, the intact type E complex, or complexes of theneurotoxin with the NBP and any one or more of the new NAPs. Suchmonoclonal antibodies can be prepared using standard hybridomatechnology (see; e.g., Kohler et al., Nature 256:495, 1975; Kohler etal., Eur. J. Immunol. 6:292, and 6:511, 1976; Hammerling et al., InMonoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981;Kruisbeck et al., Hornbeck et al., and Yokoyama, In Current Protocols inImmunology, Vol. I, New York, John Wiley & Sons, Inc., 1994). Monoclonalantibodies can be of any immunoglobulin class, including the IgG, IgM,IgE, IgA, and IgD classes, and any subclass thereof. The hybridomaproducing the mAb of the invention can be cultivated in vitro or invivo.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-7955,1984; Neuberger et al., Nature 312:604-608, 1984; Takeda et al., Nature314:452-454, 1985) by splicing the genes from a mouse antibody moleculeof appropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-426, 1988;Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; and Wardet al., Nature 334:544-546, 1989) can be adapted to produce single chainantibodies against NAPs. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide.

Antibody fragments that recognize specific epitopes can also begenerated by known techniques. For example, such fragments include, butare not limited to: the F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule and the Fab fragments that can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science 246:1275-1281, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

The binding specificity and activity of purified anti-type E (or otherserotypes) complex antibodies, such as those described above, can beconfirmed by testing their ability to interfere with the biologicalactivity of the neurotoxin and/or the complex. This ability can betested by adding the antibodies to any number of standard in vitroassays in which the release of acetylcholine from presynaptic nerveterminals can be monitored. These assays include preparations ofdifferent neuromuscular junctions, such as the mouse phrenicnerve-hemidiaphragm, the mouse plantar nerve-lumbrical muscle, and chickciliary ganglion-iris muscle preparations (Bandyopadhyay et al., J.Biol. Chem. 262:2660-2663, 1987); Bittner et al., J. Biol. Chem.264:10354-10360, 1989; Clark et al., J. Neurosci. Methods 19:285-295,1987; and Lomneth et al., Neurosci. Lett., 113:211-216, 1990). Thebinding specificity and activity of any given antibody is tested bydetermining whether that antibody effectively blocks the action of typeE neurotoxin complex applied at the neuromuscular junction.

Polypeptide-Based Detection Systems for Type E Neurotoxin AssociatedProteins

The NAPs described herein have a variety of uses, including thedetection of type E neurotoxin. The type A neurotoxin remains associatedwith its protein complex both in bacterial culture medium and in naturalcases of food poisoning (Sakaguchi, Pharmac. Ther. 19:165-194, 1983).Given this evidence, it is likely that the 118 kDa binding protein andthe other four, lower molecular weight members of the type E complexalso remain associated with the cognate toxin in vitro and in vivo. Inaddition, neurotoxin associated proteins have been shown to be moreimmunogenic than the neurotoxin itself (Singh et al., Toxicon34:267-275, 1996).

Antibodies generated against any one of the NAPs or combinations thereofcan also be used to detect the type E neurotoxin complex using variousstandard methods. For example, the antibodies can be used with a fiberoptic-based biosensor, as described herein, which uses an evanescentwave from a tapered optical fiber for signal discrimination. Thisantibody-based “sandwich” immunoassay detection system can detectbotulinum toxin much more quickly than any method currently available,but other immunoassay methods can be used. The actual signal collectionperiod with the biosensor is less than one minute. Detection isaccomplished using a two-step sandwich immunoassay. Antibody-boundoptical fibers are incubated in a solution of type E complex, and asignal is generated when the fiber-bound complex binds a fluorescentlylabeled antibody (see, Ogert et al., Anal. Biochem. 205:306-312, 1992;and Singh et al., In Natural Toxins II, B. R. Singh and A. Tu, Eds.,Plenum Press, pp. 498-508, 1996).

One of the problems historically associated with sandwich immunoassaysis that the first antibody (here, the antibody bound to the opticalfiber) and the second antibody (here, the antibody added to detect thefiber-bound complex), compete for the same epitope on the neurotoxin. Tocircumvent this problem, two antibodies can be used. The firstspecifically binds to one portion of the neurotoxin or one NAP of thetype E complex, which will be attached to the fiber, and a secondspecifically binds to either a second portion of the neurotoxin or asecond member of the polypeptide complex, which would specificallyrecognize the fiber-bound complex.

Any polypeptide (be it a NAP or an antibody of the invention) can bedetectably labeled to facilitate the detection of the type E botulincomplex. For example, the polypeptides can be conjugated with aradio-opaque or other appropriate compound, such as a fluorescentcompound, that can be brought into contact with a sample that maycontain the botulin complex. Alternatively, the polypeptide can belinked to an enzyme and used in an enzyme immunoassay (EIA; Voller etal. J. Clin. Pathol. 31:507-520, 1978; Butler, Methods Enzymol.73:482-523, 1981). The enzyme that is bound to the antibody will reactwith an appropriate substrate, e.g., a chromogenic substrate, in such amanner as to produce a chemical moiety that can be detected, forexample, by spectrophotometric, fluorimetric, or visual means. Enzymesthat can be used to detectably label a polypeptide include malatedehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeastalcohol dehydrogenase, beta-galactosidase, alkaline phosphatase,ribonuclease, and urease.

Preparation and Administration of A Neurotoxin Vaccine

The invention also includes a vaccine composition containing a type E(or other serotype) neurotoxin and more than one type E neurotoxinassociated polypeptide (or immunogenic fragment or derivative thereof)and a pharmaceutically acceptable diluent or carrier, such as phosphatebuffered saline or a bicarbonate solution (e.g., 0.24 M NaHCO₃). Thecarriers and diluents used in the invention are selected on the basis ofthe mode and route of administration, and standard pharmaceuticalpractice. Suitable pharmaceutical carriers and diluents, as well aspharmaceutical necessities for their use, are described in Remington'sPharmaceutical Sciences. An adjuvant, for example, a cholera toxin,Escherichia coli heat-labile enterotoxin (LT), or a fragment orderivative thereof having adjuvant activity, may also be included in thevaccine composition of the invention.

Skilled artisans can obtain further guidance in the preparation of avaccine for type E neurotoxin complex in Singh et al. (Toxicon27:403-410, 1990). Briefly, approximately 1.5 mg of the type E complexis added to approximately 10 ml of 0.05 M sodium citrate buffer (pH 5.5)and dialyzed against 0.39% formaldehyde at 30EC for 7 days. Theformaldehyde-containing buffer is replaced every day with fresh buffersolution. The detoxified neurotoxin (toxoid or vaccine) is dialyzedagainst 0.1 M sodium phosphate buffer (pH 7.4) without formaldehyde fortwo days with several changes of buffer.

The amount of vaccine administered will depend, for example, on theparticular vaccine antigen, whether an adjuvant is co-administered withthe antigen, the type of adjuvant co-administered, the mode andfrequency of administration, and the desired effect (e.g., protection ortreatment), as can be determined by one skilled in the art. In general,the vaccine antigens of the invention are administered in amountsranging between, for example, 1 μg and 100 mg. If adjuvants areadministered with the vaccines, amounts of the polypeptide vaccineranging between, for example, 1 ng and 1 mg can be used. Administrationis repeated as necessary, as can be determined by one skilled in theart. For example, a priming dose can be followed by three booster dosesat weekly intervals.

Treatment with Polypeptides in the Type E Neurotoxin Complex

Any disease or discomfort associated with an exaggerated release ofacetylcholine from a presynaptic nerve terminal can be treated with thepurified or isolated type E botulinum neurotoxin complex describedherein, or with complexes formed of other serotype toxins combined withone or more of the NAPS described herein. Those of skill in the art areaware of the normal parameters for acetylcholine release and of thenormal range of physiological function that is produced when anappropriate amount of this neurotransmitter is released onto a musclefrom adjacent nerve terminals. An exaggerated release of acetylcholinewould be any level of release that exceeds the normal parameters andcauses aberrant physiological function. The diseases or conditionsassociated with an exaggerated release of acetylcholine can involvespasms of either smooth or skeletal muscle cells. More specifically,these diseases or conditions include spasmodic torticollis, essentialtremor, spasmodic dysphonia, charley horse, strabismus, blepharospasm,oromandibular dystonia, spasms of the sphincters of the cardiovascular,gastrointestinal, or urinary systems, and tardive dyskinesia, which mayresult from treatment with an anti-psychotic drug such as THORAZINE® orHALDOL®.

For example, an adult male patient suffering from tardive dyskinesiaresulting from treatment with an antipsychotic drug can be treated with50-200 units (defined below) of Botulinum type E complex by directinjection into the facial muscles. Within three days, the symptoms oftardive dyskinesia, i.e., orofacial dyskinesia, athetosis, dystonia,chorea, tics and facial grimacing are markedly reduced.

Spasticity that occurs secondary to brain ischemia, or traumatic injuryof the brain or spinal cord, are similarly amenable to treatment.

In instances where the postsynaptic target is a gland, nerve plexus, organglion, rather than a muscle, the type E complex can be administeredto control profuse sweating, lacrimation, and mucous secretion. Forexample, an adult male patient with excessive unilateral sweating can betreated by administering 0.01 to 50 units of type E botulinum complex tothe gland nerve plexus, ganglion, spinal cord, or central nervoussystem. Preferably, the nerve plexus or ganglion that malfunctions toproduce the excessive sweating is treated directly. Administration oftype E neurotoxin complex to the spinal cord or brain, while feasible,may cause general weakness.

Other conditions that can be treated include tension headache and paincaused by sporting injuries or arthritic contractions. If necessary,overactive muscles can be identified with electromyography (EMG).

While it is expected that the methods of the invention will mostcommonly be applied to human patients, domestic pets (such as dogs andcats) and livestock (such as cows, sheep, and pigs) can also be treatedwith the compositions described herein.

Administration of Polypeptides within the Type E Neurotoxin Complex

The dose of type E neurotoxin complex administered to a patient willdepend generally upon the severity of the condition, the age, weight,sex, and general health of the patient, and the potency of the toxin,which is expressed as a multiple of the LD₅₀ value for the mouse.

The dosages used in human therapeutic applications are roughlyproportional to the mass of muscle to be treated. Typically, the doseadministered to the patient is from about 0.01 to about 1,000 units, forexample, about 500 units. A unit is defined as the amount of type Eneurotoxin (or type E neurotoxin complex) that kills 50% of a group ofmice (typically a group of 18-20 female mice that weigh on average 20grams). The dosage is adjusted, either in quantity or frequency, toachieve sufficient reduction in acetylcholine release to afford relieffrom the symptoms of the disease or condition being treated.

Physicians, pharmacologists, and other skilled artisans are able todetermine the most therapeutically effective treatment regimen, whichwill vary from patient to patient. The potency of botulinum toxin andits duration of action means that doses are administered on aninfrequent basis. Skilled artisans are also aware that the treatmentregimen must be commensurate with questions of safety and the effectsproduced by the toxin.

Typically, the type E neurotoxin complex is suspended in aphysiologically acceptable solution, such as normal saline, and isadministered by an intramuscular injection. Prior to injection, carefulconsideration is given to the anatomy of the muscle group, in an attemptto inject the toxin complex into the area with the highest concentrationof neuromuscular junctions. If the muscle mass is not very great, theinjection can be performed with extremely fine, hollow, teflon-coatedneedles and guided by electromyography. The position of the needle inthe muscle should be confirmed prior to injection of the toxin, andgeneral anesthesia, local anesthesia, or other sedation may be used atthe discretion of the attending physician, according to the age andparticular needs of a given patient and the number of sites to beinjected.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES

Production of Botulinum Toxin by Cell Culture

Generally, to obtain botulinum toxin in large amounts, complexed mediacontaining combinations of meat hydrolysate, casein hydrolysate, yeastautolysate, yeast extract, and glucose supplemented with one or morereducing agents are used (Sakaguchi, Pharmac. Ther. 19:165-194, 1983).Vegetables autoclaved in saline also provide an excellent culturemedium, supporting toxin production by type A- and type B-producingbacteria to a similar extent as laboratory media. Glucose must be addedfor type E- and type F-producing bacteria to grow in boiled vegetables(Sugii et al., J. Food Safety 1:53-65, 1977). The optimum temperaturefor toxin production by C. botulinum is generally regarded as 20-35° C.

Type E C. botulinum Produces a Complex Including Five NeurotoxinAssociated Proteins

For this series of experiments, C. botulinum type E (available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.,20852 (U.S.A.); Type E Clostridium botulinum Accession Nos. 9564, 17786,17852, 17854, and 17855) was grown for 4 days in 15 ml cooked meatmedium. Stock cultures were prepared according to standard methods andstored at −20° C.

The stock culture was activated at 30° C. for approximately 25 hours andthen transferred to a growth medium containing 2.0% Trypticase-peptone,1.0% glucose, 0.025% sodium thioglycolate (BBL Microbiology Systems,Cockeysville, MD), and 0.5% yeast extract (Difco) adjusted to pH 6.5.When large culture volumes (8 liters) were used, a 12% glucose solutionwas autoclaved and added to the broth, which was separately prepared andthen autoclaved for 1 hour. The culture was incubated for 60-65 hours,and cells were collected by centrifugation. An extract from the cellswas prepared at 20EC by stirring with 0.2 M phosphate buffer (pH 6.0).The resulting suspension was saturated with (NH₄)₂SO₄; 39 g/ml) andstored at 4EC.

DEAE-Sephadex Chromatography

The crude extract described above was precipitated and redissolved in 35ml of 0.05 M sodium phosphate buffer (pH 5.5). The resulting solutionwas clarified by centrifugation and chromatographed on a DEAE-SephadexA-50 ion-exchange column (Pharmacia). The sample was eluted from thecolumn with 0.05 M sodium citrate at pH 5.5. It is important that the pHof the buffer is maintained at 5.5. The first protein peak (FIG. 1) waspooled as type E complex.

In contrast to previous reports (Sugii et al., Infect. Immunol.12:1262-1270, 1975; Sakaguchi, Pharmac. Ther. 19:165-194, 1983; Schantzet al., Microbiol. Rev. 56:80-99, 1992; Singh et al., J. Protein Chem.14:7-18, 1995), a total of five different proteins were found in thecomplex in addition to the 150 kDa type E botulinum neurotoxin.Specifically, the material constituting the first peak of the elutionprofile described above (and shown in FIG. 1) was analyzed bySDS-polyacrylamide gel electrophoresis. Six proteins, having molecularweights of approximately 150 (the neurotoxin), 118, 80, 65, 40, and 18kDa, were apparent (FIG. 2).

Size Exclusion Chromatography

To further confirm the nature of the type E complex, the proteins wereanalyzed on size exclusion chromatographic columns. The type E complexthat eluted from the DEAE Sephadex A-50 column was concentrated to 5mg/ml and applied to a Sephadex G-100 column (1.8×92 cm or 2.6×82 cm,0.05 M sodium citrate buffer, pH 5.5). The resulting elution profilerevealed one peak in the void volume (FIG. 3). Three of the proteinspresent are clearly less than the exclusion limit of the column andthus, should elute separately from the void volume. Since this did notoccur, and all six proteins continued to elute in one peak, it wasconcluded that the proteins are bound together in a complex. A similarresult was obtained following chromatography on a Sephadex G-200 column(FIG. 4), further confirming that the six proteins form a singlecomplex.

One of the neurotoxin associated proteins in the type E complex, the 80kDa protein, was purified and studied for its ability to re-form acomplex with pure type E neurotoxin. After combining the 150 kDaneurotoxin and the 80 kDa associated protein, the elution profileobtained from a Sephadex G-200 column revealed a major peak containingboth the 80 kDa protein and the type E neurotoxin, and a minor peakcontaining excess uncomplexed 80 kDa protein (FIG. 5). The materialeluted in each of the two peaks was electrophoresed on anSDS-polyacrylamide gel (FIG. 6), which confirmed the content of theprotein(s) in each peak.

The 80 kDa Neurotoxin Associated Protein Specifically Binds Type ENeurotoxin

A kinetic binding study performed with an optical fiber-based biosensorrevealed that the type E neurotoxin could bind directly to the 80 kDatype E neurotoxin associated protein, rather than associate indirectlywith the neurotoxin via another polypeptide in the complex. The 80 kDapolypeptide was tested for its ability to bind directly to theneurotoxin at pH 7.5 and at pH 5.7. The type E botulinum neurotoxin wasfirst immobilized, and the purified 80 kDa NAP was labeled with TRITC(Tetramethyl-rhodamine-isothiocyanate; Molecular Probes, Eugene, Oreg.)as described in Ogert et al. (Anal. Biochem. 205:306-312, 1992), exceptthat unreacted TRITC was removed by dialysis, rather than gelfiltration. The binding experiments were carried out by blocking theexposed sites on the optical fiber with 2% BSA (at room temperature) andincubating them with TRITC-labeled 80 kDa polypeptide (5 mg/ml) that hadbeen equilibrated with phosphate buffered saline (PBS; at pH 5.7 or pH7.5). The initial rate of binding was calculated based on the signalincrease within the first 60 seconds.

Subsequent polypeptide binding rates at pH 7.5 and 5.7 were 4.01 and8.42 μV/minute, respectively, suggesting that the interaction betweenthe neurotoxin and the 80 kDa polypeptide is significant at pH 7.5, andconsiderably higher at pH 5.7. Therefore, the 80 kDa polypeptide couldplay a role in protecting the type E neurotoxin from the acidicconditions present in the gastrointestinal tract.

These results are consistent with the known behavior of the botulinumneurotoxin complex, which dissociates at alkaline pH levels. Thus, theassociated binding polypeptides can be used as a specific bindingpartner to “capture,” and thereby detect, the neurotoxin. This methodwould effectively detect the neurotoxin wherever it exists, to at leastsome degree, free from the complex or, at least, free from the 80 kDaneurotoxin binding protein. The 80 kDa NAP also complexes with Type Aneurotoxin.

Sequence Analysis of Proteins in the Type E Neurotoxin Complex

Partial amino acid sequences of the novel polypeptides in the serotype Eneurotoxin complex were obtained as follows. Approximately 10 picomolesof the purified type E neurotoxin complex were dissolved in a bufferconsisting of 0.5 M sucrose, 15% SDS (sodium dodecyl sulfate), 312.5 mMTris, and 10 mM EDTA, and electrophoresed on a 12.5% SDS-acrylamide gelusing a Mini-PROTEAN II™ electrophoresis cell (Bio-Rad Laboratories,Hercules, Calif.). The electrophoresis was performed in running buffer(2 g/L Tris base, 14.4 g/L glycine, 1 g/L SDS and 0.1 mM sodiumthioglycolate, pH 8.3) under a constant voltage (200 V). The protein wasthen electrotransferred from the gel to a PVDF membrane in a Twobinbuffer (25 mM Tris, 192 mM glycine and 20% methanol) using a MiniTrans-Blot electrophoretic transfer cell™ (Bio-Rad Laboratories,hercules, Calif.). The transfer was carried out overnight at 60 volts inan ice bath. To visualize the protein bands, the membrane was stainedwith 0.025% Coomassie Blue 8250 in 40% methanol and destained with 50%methanol. The proteins bound to the PVDF membranes were sequenced atBaylor College of Medicine (Houston, Tex.) using Applied Biosystem Model473A protein sequencer™ (Foster City, Calif.).

The following peptide sequences were obtained: (1) MKQAFVFEFD (SEQ IDNO:1), from the 18 kDa protein; (2) MRINTNINSM (SEQ ID NO:2), from the40 kDa protein; (3) MQTTTLNWDT (SEQ ID NO:3), from the 65 kDa protein;and (4) TNLKPYIIYD (SEQ ID NO:4), from the 80 kDa protein. Thesesequences were compared with those of known proteins associated withneurotoxin types A, B, and C. This analysis failed to reveal any regionsof homology with the type A associated proteins of C. botulinum.

Genomic Organization

The genomic organization of NAPs of type E C. botulinum was investigatedusing oligonucleotide primers having sequences complementary to theN-terminal amino acid sequences. Using combinations of sense andantisense primers (the sequences of which were derived from the aminoacid sequences shown in SEQ ID NOs:1-4 using standard techniques) in aconventional polymerase chain reaction (where chromosomal DNA frombacteria of the genus Clostridia served as the template), the topographyof the 18 and 80 kDa NAPs were revealed. Both of these genes aretranscribed in the opposite direction to that of the neurotoxin and NBP(i.e., the 118 kDa neurotoxin binding protein) genes (FIG. 9). Inaddition, there is an open reading frame between the NBP and the 18 kDaNAP that has the same transcriptional direction as the NBP. This proteinsequence did not match with any of the proteins that have beenidentified as NAPs of type E C. botulinum. In addition to revealing theorientation of various NAP-encoding genes, these experiments revealedthat the genes are clustered together within the genome.

Analysis of Type E Neurotoxin Complex by Light Scattering

To characterize the type E neurotoxin complex as a whole, lightscattering experiments were performed on material purified byDEAE-Sephadex A-50 chromatography (1.5 mg/ml). Analysis was performed ona Malvern 4700 PCS Autosizer System (Malvern Instruments Inc.) equippedwith an eight-bit, 136 channel correlator capable of variable timeexpansion. The laser light source was model INNOVA 70-5 argon laser(Coherent, Calif.). A 514.5 nm line was employed in single operationmode with 1.0 watt power output.

Initial results from light scattering experiments suggested that thecomplex exists in two forms, as 600 and 2000 kDa molecular weightspecies (FIG. 7). The combined molecular weight of the proteins in thetype E neurotoxin complex observed on polyacrylamide gels is 468 kDa.The difference between these two predicted sizes could be due either tovariation in the folding of the complex or to the existence ofoligomeric forms of some of the proteins in the complex.

NAPs Protect the Toxic Activity of Botulin Neurotoxin from Heat

To investigate the role of NAPs in protecting the type E botulin complexfrom heat, the neurotoxin alone and the intact complex were each heatedto 60EC for 15 minutes, then cooled, and incubated with “cracked” PC12cells that had been grown in tritiated norepinephrine and stimulated torelease norepinephrine by calcium. The term “cracked” is used todescribe cells that have been treated with a mechanical device so thattheir integrity is somewhat disrupted; this technique is described inLomneth et al., J. Neurochem. 57:1413-1421 (1991). The percentage ofnorepinephrine released into the culture medium was then assessed,because the toxin blocks release of norepinephrine from the crackedcells.

In control experiments, the neurotoxin alone and the intact complex wereeach incubated with cracked PC12 cells, but not subjected to the 60ECheat treatment. The control botulin neurotoxin complex (i.e., a complexthat had not been heat-treated) reduced the percentage of norepinephrinerelease from a normal 56.1∀0.8% in buffer-treated (no complex, no toxin)cells, to 22.5±0.3% in cells treated by the neurotoxin complex at afinal concentration of 50 μg/ml.

In spite of heat treatment (and potential denaturation), theheat-treated complex, at the same concentration of 50 μg/ml, was stillable to block, i.e., reduce, the percentage of norepinephrine release to34.1±0.9%. The type E neurotoxin, when not heated and used alone (i.e.,without NAPs) blocked (or reduced) the percentage of norepinephrinerelease to 21.0±0.6% at 50 μg/ml, whereas heat-treated neurotoxin wasnot able to substantially block the norepinephrine release (the percentof norepinephrine released was 51.9±1.9%). These data clearly suggestthat the presence of NAPs is effective in providing functional stabilityto the type E botulinum complex.

To determine whether the NAPs provided protection to the botulin complexby preventing heat-induced unfolding of polypeptides in the complex, orby assisting in proper refolding of heated toxin, the heat-inducedunfolding of purified neurotoxin and the neurotoxin complex wereanalyzed by monitoring their circular dichroism (CD) signal at 222 nm.This method has been used successfully to monitor unfolding of proteins(see, e.g., Fahnestock et al., Science 258:1658-1662, 1992; and Lehrerand Qian, J. Biol. Chem. 265:1134-1138, 1990). The midpoint unfoldingtemperature (Tm) for the neurotoxin was 54EC whereas the Tm for thelarge (new) type E complex was 70EC. The Tm for a neurotoxin complexconsisting of the 118 kDa NBP and the type E botulin neurotoxin isbetween these two values (Sakaguchi, Pharmac. Ther. 19:165-194, 1983;Singh et al., J. Protein Chem. 14:7-18, 1995). These observationsclearly indicate that the loss of type E botulin neurotoxin activityafter heating at 60EC is due to the unfolding of the toxin, whereas nosuch unfolding occurs in the presence of NAPs.

An observation made while conducting this set of experiments was thatthe type E botulinum complex is equally (or more) effective in blockingthe neurotransmitter release from PC 12 cells compared to the pureneurotoxin (without NAPs), although the total effective concentration ofthe neurotoxin in the complex was only about a third of the pure toxinconcentration (111 nM vs. 333 nM). This suggests that the NAPs actuallyactivate the neurotoxin, which would be consistent with our hypothesisthat the folding of the type E complex can be altered by NAPs.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, that the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A substantially pure polypeptide complex comprising a Clostridium botulinum neurotoxin and more than one Clostridium botulinum type E neurotoxin associated polypeptide.
 2. A complex of claim 1, wherein the neurotoxin associated polypeptide has a molecular weight of approximately 80 kDa and comprises the amino acid sequence TNLKPYIIYD (SEQ ID NO:4).
 3. A complex of claim 1, wherein the neurotoxin associated polypeptide has a molecular weight of approximately 65 kDa and comprises the amino acid sequence MQTTTLNWDT (SEQ ID NO:3).
 4. A complex of claim 1, wherein the neurotoxin associated polypeptide has a molecular weight of approximately 40 kDa and comprises the amino acid sequence MRINTNINSM (SEQ ID NO:2).
 5. A complex of claim 1, wherein the neurotoxin associated polypeptide has a molecular weight of approximately 18 kDa and comprises the amino acid sequence MKQAFVFEFD (SEQ ID NO:1).
 6. A complex of claim 1, wherein the neurotoxin associated polypeptide has a molecular weight of approximately 18 kDa and comprises the amino acid sequence shown in FIG. 8 (SEQ ID NO:5).
 7. A substantially pure Clostridium botulinum serotype E neurotoxin associated polypeptide.
 8. The polypeptide of claim 7, wherein the neurotoxin associated polypeptide has a molecular weight of about 80 kDa.
 9. The polypeptide of claim 8, wherein the neurotoxin associated polypeptide comprises the amino acid sequence TNLKPYIIYD (SEQ ID NO:4).
 10. The polypeptide of claim 7, wherein the neurotoxin associated polypeptide has a molecular weight of about 65 kDa.
 11. The polypeptide of claim 10, wherein the neurotoxin associated polypeptide comprises the amino acid sequence MQTTTLNWDT (SEQ ID NO:3).
 12. The polypeptide of claim 7, wherein the neurotoxin associated polypeptide has a molecular weight of about 40 kDa.
 13. The polypeptide of claim 12, wherein the neurotoxin associated polypeptide comprises the amino acid sequence MRINTNINSM (SEQ ID NO:2).
 14. The polypeptide of claim 7, wherein the neurotoxin associated polypeptide has a molecular weight of about 18 kDa.
 15. The polypeptide of claim 14, wherein the neurotoxin associated polypeptide comprises the amino acid sequence MKQAFVFEFD (SEQ ID NO:1).
 16. The polypeptide of claim 14, wherein the neurotoxin associated polypeptide comprises the amino acid sequence shown in FIG. 8 (SEQ ID NO:5).
 17. A substantially pure antibody that specifically binds to a Clostridium botulinum type E neurotoxin associated polypeptide having a molecular weight of approximately 80, 60, 45, or 18 kDa, or to a complex of any two or more of said neurotoxin associated polypeptides.
 18. A substantially pure antibody that specifically binds to a polypeptide complex of claim
 1. 19. A method of detecting a serotype E neurotoxin complex in a sample, the method comprising: (a) contacting the sample with an antibody of claim 17, and (b) detecting antibody-bound polypeptide, if any, in the sample, the presence of antibody-bound polypeptide indicating the presence of serotype E neurotoxin in the sample.
 20. The method of claim 19, wherein the sample is a foodstuff.
 21. The method of claim 19, wherein the sample is a gastrointestinal, blood, or tissue sample obtained from a vertebrate animal.
 22. A method of treating a patient who is suffering from a disease or condition associated with excessive release of acetylcholine from presynaptic nerve terminals, the method comprising administering to the patient a therapeutically effective amount of a polypeptide complex of claim
 1. 23. The method of claim 22, wherein the excessive acetylcholine release causes undesirable contraction of smooth or skeletal muscle cells.
 24. The method of claim 22, wherein the excessive release of acetylcholine causes profuse sweating, lacrimation, or mucous secretion.
 25. A method of treating a patient who is suffering from spasticity occurring secondary to brain ischemia, or traumatic injury of the brain or spinal cord, the method comprising administering to the patient a therapeutically effective amount of a polypeptide complex of claim
 1. 26. A method of treating a patient who is suffering from tension headache or pain, the method comprising administering to the patient a therapeutically effective amount of a polypeptide complex of claim
 1. 27. A vaccine comprising a polypeptide complex of claim
 1. 28. A method of vaccinating an animal against serotype E neurotoxin, the method comprising administering to the animal an effective amount of the vaccine of claim
 27. 29. A vaccine comprising a polypeptide of claim
 7. 30. A method of detecting a Clostridium botulinum serotype E neurotoxin in a sample, the method comprising: (a) contacting the sample with a Clostridium botulinum type E neurotoxin associated polypeptide (NAP) of claim 7 that specifically binds a serotype E botulinum neurotoxin and thereby forms a NAP-neurotoxin complex, and (b) detecting the NAP-neurotoxin complex, if any, in the sample, the presence of a complex indicating the presence of serotype E neurotoxin in the sample.
 31. A complex of claim 1, comprising the neurotoxin and neurotoxin associated polypeptides having molecular weights of about 80 kDa, 65 kDa, 40 kDa, and 18 kDa.
 32. The complex of claim 1, comprising the neurotoxin and neurotoxin associated polypeptides having molecular weights of about 118 kDa, 80 kDa, 65 kDa, 40 kDa, and 18 kDa. 